Inflammatory diseases are characterized by movement of leukocytes into affected tissues. Once there, cells such as polymorphonuclear leukocytes (PMN) are thought to cause tissue damage directly through the release of lytic enzymes and reactive small molecules such as superoxide anion, and indirectly by release of inflammatory mediators. Depletion of PMN from the circulation of animals minimizes tissue damage in several inflammatory settings.
The CD18 antigens are a family of three heterodimeric receptors sharing a common .beta. chain, CD18, and homologous but distinct .alpha. chains, CD11a, CD11b and CD11c. These receptors are expressed on all leukocytes, and are members of the integrin superfamily (R. O. Hynes, 1987). Integrins are .alpha..sub.1 .beta..sub.1 heterodimers that require divalent cations and warm temperatures for ligand binding (Hynes, Supra.) and mediate cell-cell and cell-extracellular matrix adhesion interactions. Many of the ligands are bound via a tripeptide sequence, Arg-Gly-Asp, or RGD. The leukocyte integrins (B.sub.2 integrins or CD18 antigens) participate in adhesion of cells by recognizing a variety of surface bound ligands including ICAM-1, ICAM-2, C3bi, fibrinogen and unidentified structures on endothelium (S. D. Wright et al., 1990).
One of the CD18 antigens, complement receptor type 3 (CR3, also known as CD11b/CD18), is found on human polymorphonuclear leukocytes (PMN), monocytes and macrophages. Ligands for this receptor include C3bi, a cleavage product of the third component of complement, fibrinogen, and an as yet uncharacterized molecule on endothelial cells (S. D. Wright et al., Supra.). A second type of ligand is the lipid IVa portion of lipopolysaccharide from gram-negative bacteria (S. D. Wright et al., 1986; S. D. Wright et al., 1989) which binds at a site on the receptor that can be distinguished from that for the proteinaceous ligands by blockade with distinct monoclonal antibodies.
Movement of PMN into tissues can be dramatically reduced by blocking CD18 antigens with monoclonal antibodies. Such blockade has been found to prevent PMN migration into the peritoneal cavity, brain, heart, bowel and skin and has been shown to reduce tissue injury in models of meningitis, cardiac and bowel ischemia and reperfusion injury and hemorrhagic shock (E. I. Tuomanen et al., 1989; P. J. Simpson et al., 1988; N. B. Vedder et al., 1988; L. A. Hernandez et al., 1987). This course of therapy has been suggested in U.S. Patent No. 4,797,277 to Arfors with respect to reperfusion injury, and in European Patent Application No. 0 346 078 A2 published 13 December, 1989, With regard to a variety of inflammatory conditions related to PMN migration.
In both publications, certain antibodies were identified and postulated to block the CD18 receptor and consequently, the migration and adhesion of PMN to endothelium. The structure and origin of these antibodies, however, did not suggest an endogenous intracellular mechanism to account for the restriction of CD18 function to sites of inflammation.
The capacity of the CD18 molecule to bind endothelium (or any of its ligands) may be rapidly turned on and off. This enables leukocytes to rapidly adhere to endothelium upon stimulation by chemoattractants, and further allows cells to locomote by adhering at their leading edge and releasing adhesion at the uropod. This regulated behavior enables CD18 to promote movement of leukocytes into tissues.
CR3 on human PMN binds its ligands in a regulated manner. This property was first described by Wright and Meyer (S. D. Wright et al., 1986) using a rosetting assay. PMN are first allowed to adhere to a culture well, and sheep erythrocytes bearing covalently attached C3bi on their surface (EC3bi) are then added. After a brief incubation, unbound E are washed away and the number of E bound per 100 PMN is scored by phase contrast microscopy (the attachment index). Visually, the smaller E form a rosette-like arrangement when attached to PMN.
CR3 on resting PMN binds poorly to its ligand, C3bi; however, when the PMN are treated with agonists such as phorbol myristate acetate (PMA), the binding activity of CR3 transiently increases. After 20 minutes in PMA, rosetting rises by 5-10 fold, but by 60 minutes in PMA the rosetting declines below starting levels. The change in binding of C3bi is only partially explained by the observed 2-3 fold increase in surface receptor numbers that are released from specific granules. Receptor expression remains high after degranulation, even though binding falls back down below baseline.
In addition, studies (S.K. Lo et al., 1989) show CD18 activity is enhanced after phorbol ester stimulation of PMN-derived cytoplasts, which lack specific granules and therefore have no means of increasing receptor numbers at the plasma membrane. Therefore, some other means of regulation is involved in the transient changes in binding. Detmers et al. (1987) have shown that enhanced receptor binding correlates with the localization of receptor in small clusters on the cell surface. However, the mechanism of such clustering is unknown, as is any effect on the K.sub.d of individual receptors in the cluster, and it is toward the elucidation of these phenomena that the present invention is generally directed.